This project, directed toward elucidation of the molecular mechanisms responsible for cataractogenesis and the development of means of prevention of this disease, places special emphasis on the structure and function of t lens crystallins and the role these proteins play in lens opacification. U il recently, crystallins were thought to be simply structural elements of the lens matrix without specific quantifiable biological functions. Two recent discoveries have provided new insights and approaches to the physiological roles of the crystallins: (1) the crystallins are either functionally activ enzymes or are at least related to proteins with specific biological activi es, and (2) alpha-crystallin is a molecular chaperone that can prevent the aggregation of denaturing proteins. We believe that crystallins have specific biological functions in addition their structural role in forming the transparent lens tissue. The stronges example to date is the chaperone-like function of `-crystallin. This major lens protein prevents the aggregation of other proteins that are undergoing modification and denaturation. In the lens, where protein turnover is extremely limited and where the long-lived crystallins are known to undergo extensive structural modification, alpha-crystallin may be essential in preventing protein aggregation and precipitation. Such precipitation would destroy the optical transparency of the lens by creating light scattering centers. We have also developed evidence that the enzyme/crystallins are contributing to the antioxidative capacity of the lens, primarily by marked increasing the pool of reduced pyridine nucleotides in the lens. We can demonstrate the utilization of the nucleotide's reducing capacity in eliminating activated species of oxygen generated by Fenton chemistry or by other mechanisms. Further support for the view that enzyme/crystallins have specific functions was obtained from studies on the expression of zeta crystallin in the lenses of guinea pigs and llamas. The data clearly demonstrate that the zeta-crystallin gene was recruited by the lens independently in each of the two species. This finding strongly supports a selective basis for the recruitment rather than a neutral evolution mechanism and indicates that the protein must provide significant benefit to the lens of these species. Diabetic retinopathy is the major cause of blindness in adults (20 to 74 years old) in the industrialized countries. Whereas systemic diabetes mellitus results from lowered availability and/or cellular recognition of insulin, the complications of diabetes, such as diabetic retinopathy, are caused by the chronic hyperglycemia itself. Exaggerating the hyperglycemic effect by feeding rats a galactose diet, we produced the first rat model fo diabetic retinopathy. Intervention studies designed to simulate clinical t als were used to test the possibility of delaying, halting, or reversing retinopathy soon after the earliest capillary lesions could be documented. Weanling male Sprague-Dawley rats were divided into 10 groups, 4 of which received either normal lab chow or a 50 percent galactose diet with or without one of two aldose reductase inhibitors (ARIs: AL-3152 or WAY- 121,509), and other groups that received 50 percent galactose for 4, 6, or 8 months and then intervention either by addition of inhibitor or removal of galactose. From rats killed at 4, 6, 8, 16, 18, and 24 months, one reti was prepared for obtaining electron micrographs of capillary transections; the other was used for whole mounts of isolated retinal vessels. Images of whole and transected capillaries were captured and analyzed using computer hardware and programs specially designed for 1024-x 1024-x 8-bit resolution Based on several quantitative assessments, including basement membrane thickness, PAS stain intensity, cellularity, dilation, tortuosity, length, d microaneurysm, the retinopathy was graded on a scale of 0 to 10. At 6 months, when intervention was begun, the untreated galactose-fed rats exhibited a 30 percent, statistically significant (p<0.01) increase in capi ary basement membrane thickness and grade 1 retinopathy overall. By 18 months, the same group had grade 7 retinopathy, whereas the rats receiving intervention with an ARI-enriched or galactose-free diet exhibited only grade 2 retinopathy, and the rats fed control diet or galactose plus AL-315 throughout the 18 months showed no retinopathy. At 24 months, the untreated rats exhibited grade 10 retinopathy, and both intervention groups had a grade 8.5 retinopathy. In conclusion, intervention at 6 months delay but does not halt or reverse the progression of galactose-induced retinopathy. We plan to attempt, by dietary manipulation, to produce rat models that develop the diabetic-like retinal angiopathies sooner. Also, us g cell and organ culture, we will investigate the possible mechanisms.